Posted December 17, 2010
The ability to detect circulating tumor cells (CTCs) in a whole blood specimen has the potential to significantly influence the practice of pathology. Its application includes risk (prognosis) stratification of cancer patients, early detection of relapse, response monitoring to treatment in patients with metastatic carcinoma, and if the CTCs can be acquired for phenotypic and/or genotypic analysis, they can potentially improve therapy selection and develop novel targeted therapies.
CTCs gained visibility when the FDA approved the Veridex CellSearch Epithelial Cell Kit/CellSpotter Analyzer in January 2004 for the enumeration of CTCs of epithelial origin in whole blood. In December 2006, a modification to the intended use allowed the test to be used as an aid in the monitoring of patients with metastatic breast cancer. In 2007, the clearance was expanded to include metastatic colon cancer and in 2008, metastatic prostate cancer. CTC technology has been around for more than a decade and several image analysis systems are FDA-cleared to detect rare events such as CTCs on immunostained slides.
CTCs can be detected by either microscopy or polymerase chain reaction (PCR) in addition to Veridex system. PCR is more sensitive than microscopy for detecting the presence of CTCs but is less specific and more importantly, does not allow for the capture or visualization of the detected cells, to date. The use of microscopy typically requires an enrichment step, which concentrates the CTCs into a fraction that can then be transferred to a slide or other detection medium. Enrichment is currently performed by filtration, density gradient ultracentrifugation, or a variety of antibody separation techniques. Once the specimen is placed on a slide and stained, an image analysis system scans the entire slide and presents images for the pathologist to review. Based on morphology and staining characteristics, the pathologist enumerates the tumor cells.
Currently, CTCs are a measure of therapy efficacy but pathologists may be able to influence chemotherapy choices if CTCs can be isolated and genotyped. Potentially, CTCs could be analyzed for all carcinomas repeatedly: initially as part of a preoperative workup to establish the baseline, then peri- (or even intra-) operatively to assess adequacy of excision, and then still again post-operatively at sequential time intervals as part of monitoring for early relapse. Moreover, any of these CTC evaluations could also direct and modify therapy choices. Several publications show differences in the genotype and phenotype of primary tumors and their circulating tumor cells.
CTC technology is likely to become integral in providing personalized care to cancer patients and could significantly expand the role of the pathologist. Instead of performing prognostic and predictive testing only once at the time of diagnosis, genotyping and/or phenotyping of CTCs has the potential to monitor and adjust therapy choices at defined intervals, turning an isolated diagnostic event into a program of serial assessments as the management of cancer converts an acute disease into a chronic medical management process. Pathologists are well positioned to combine their anatomic and clinical pathology skills, implement the new technologies, supervise and train technologists and possibly benefit from increased diagnostic opportunities.
Most of the current focus on CTCs has been to enumerate them as a monitoring tool to measure the success of therapy and prognosis stratification. Because the current enrichment and detection systems lack sensitivity, the inability to demonstrate CTCs does not mean that they are not present. However, if the system is sensitive enough to detect CTCs in a patient, then that same methodology can be used to monitor the number of CTCs in that patient over time.
Other uses may predominate in the future. Several studies have identified CTCs as an independent predictor of prognosis in breast, prostate and colorectal carcinoma and the ability to isolate and analyze CTCs has the promise to be more predictive of response to therapy. Although the studies are limited, some primary breast carcinomas classified as HER-2 negative by FISH have demonstrated HER-2 gene amplification in their CTCs and, more importantly, many have responded to anti-HER-2 therapy. Similarly, some lung carcinomas lacking detection of an EGFR mutation in their primary carcinoma have circulating tumor cells with detectable mutations. These patients respond to tyrosine kinase inhibitors, as would be expected in tumors demonstrating an EGFR mutation. In the future, we may be assessing prognostic and predictive factors on CTCs rather than primary carcinomas.
At present, the application of this technology has not been sufficiently proven to affect outcomes in patients with metastatic cancer, at least in the opinion of most insurers and the latest ASCO recommendation for the use of tumor markers in breast cancer. This is based on the lack of peer-reviewed medical literature indicating that CTCs can be used to alter therapy decisions and improve outcome. The reviewers stated that while CTCs have potential for use in patient monitoring, there is currently insufficient data to determine if this technology is effective as a marker of disease progression or even if it is better than existing tumor markers in terms of efficacy and clinical utility.
The technologies employed to detect and enumerate CTCs are varied but the underlying principles are similar. The first step is sample enrichment for epithelial cells. This is achieved by exploiting the size differences between tumor cells and blood cells, or by antigenic differences of epithelial cells. Ultrafine filters can be used to capture the relatively larger tumor cells while allowing the blood cells to pass through the filter unobstructed. After washing, the captured epithelial cells are released from the filters. The efficiency of filters varies with the size differences, and can fail if the tumor cells are small, such as with some lobular breast carcinomas and neuroendocrine carcinomas. Density gradient centrifugation can also enrich samples based on physical properties. A commercial product, OncoQuick®, consists of a sterile 50 ml Polypropylene tube with a porous barrier inserted on top of a proprietary separation medium, but as with ultrafine filters, limitations may exist for some cell types.
Antigenic differences can also aid in sample enrichment. Immunomagnetic separation techniques generally utilize antibodies attached to iron particles with the antibody either recognizing epithelial cells in the case of positive separation or white blood cells in the case of negative separation. Several problems limit the efficiency of the positive separation technique. First, the antibody used must recognize and bind to the tumor cell. There is no universal antibody that identifies all carcinoma cells without cross-contamination from some unwanted cells. In the case of CellSearch, the iron particles are associated with an EpCam antibody to identify most (but not all) breast cancer cells. Once the cell of interest is bound to the antibody, a magnetic field separates the antibody-iron particle complex from the unrecognized cells. The epithelial cell must then be released from the iron particle; another step with less than 100% efficiency. When normal blood was spiked with a breast cancer cell line known to react with the EpCam antibody, only 85% of the spiked cells were recovered. However, systems designed to monitor CTCs over time are more concerned with consistency than with overall efficiency and do not claim to be able to detect all circulating tumor cells.
Negative separation techniques may also be used. The chosen antibody identifies white blood cells (typically via CD45) and the magnetic field is used to separate the unwanted cells, leaving the cells of interest behind. On face value, this may seem like a better technique since it does not depend upon the release from the antibody, however in practice, epithelial cells can be physically surrounded and entrapped by the antibody-iron complex and dragged out of the mixture when the magnet is applied.
A recent publication described a microfluidic-based device, named a CTC-chip that can isolate, quantify, and analyze circulating tumor cells from blood samples. Blood flows past 78,000 EpCAM-coated microposts under controlled conditions that optimize the capture of circulating tumor cells. An average of 132 circulating tumor cells per milliliter was isolated at high purity from patients with metastatic cancers but not from healthy controls demonstrating the efficacy of the technique.
Once epithelial cells are separated, they must be identified by the pathologist. A cytospin preparation is typically used to produce the slide and then the slide is stained to aid in the detection of the tumor cells. Although the specimen is enriched, there may still be no or few epithelial cells present. In the case of CellSearch a mixture of two phycoerythrin-conjugated antibodies that bind to cytokeratins 8, 18, and 19; an antibody to CD45 conjugated to allophycocyanin; and the nuclear dye 4",6- diamidino-2-phenylindole (DAPI) is applied to fluorescently label the cells. Some researchers stain the cytospin slide with a pancytokeratin antibody and detect the signal chromogenically, usually utilizing fast red detection. In either case, an image analysis system is employed to enumerate the cells, since such systems have shown a marked improvement in sensitivity and reproducibility. The image analysis system scans the slide at high power magnification and acquires images of each cell on the slide. Depending on the image analysis system, either all of the identified cells or only cells meeting predefined criteria are displayed to the pathologist for identification as a tumor cell. CellSearch identifies each cell for the pathologist as a montage of 4 images: the composite image, the DAPI image, the phycoerythrin channel image and the allophycocyanin channel image. An epithelial cell is defined as a cell with a round to oval morphology, a visible nucleus (DAPI positive), cytokeratin positive and CD45 negative. This is a relatively time consuming process for the pathologist and requires some specialized training. Chromogenic image analysis systems present only those cells showing some level of red expression (if fast red detection is used), but such systems cannot distinguish real staining from artifact. Each cell is displayed to the pathologist who, based on the morphology and staining pattern, classifies it as artifact, normal cell or tumor cell. Similarly, this can be a time consuming process for the pathologist.
As with all tests, the performance characteristics must be established before reporting results. To date, the performance characteristics of the CellSearch assay have been best studied. The linearity of the system was assessed in patients using spiked cell lines and was shown to be linear over a range of 5 to 1142 cells with an average recovery of 85% of the cells. Only 1 of 344 healthy volunteers and patients with non-malignant disease showed greater than or equal to 2 CTCs per 7.5 ml of blood. In patients with known metastatic disease 37% (489 of 1316) of breast cancer patient, 57% (107 of 188) prostate cancer patients and 30% (99 of 333) colorectal cancer patients demonstrated greater than or equal to 2 CTCs.
Currently, there is only a single vendor of a complete system although several vendors make components that could be put together in a laboratory-developed assay. Veridex CellSearch is an FDA cleared complete commercial system which includes hardware, software and staining kits. The system requires significant capital expense and training. Greiner BioOne manufactures sterile 50 ml Polypropylene tubes with porous barriers on top of a proprietary separation medium. It works based on density centrifugation and purports to enrich blood samples with efficiency similar to immunomagnetic separation. Several companies manufacture immunomagnetic bead separation columns, supporting both positive and negative separation. There are also several vendors of image analysis systems capable of rare event detection which is necessary if one is using a chromogenic detection system.
Oncologists predominantly, with increasing frequency, are requesting CTCs. New England Journal of Medicine, Journal of Clinical Oncology and other leading journals have reported on the efficacy of such systems. However, ASCO or most insurers have not endorsed CTC detection as they still classify the procedure as experimental. The test must be performed in a pathology laboratory but because of the wide variety of methodologies employed, it is frequently unclear in which section of pathology the instrument should be placed. Components of the assay could typically be run in hematology, cytology, immunohistochemistry, image analysis and chemistry sections. This diversity of components is likely one of the main barriers to the development of laboratory developed assays (LDTs).
In the future, CTCs could have a major impact to pathologists:
- Clinical Impact: Currently we diagnose a tumor once and perform prognostic/predictive testing at that time. CTCs have the potential to turn the management of cancer patients into a continuous specimen stream with analysis, pre- and post- surgical resection and then continuous CTC testing to monitor for early recurrence in negative patients and to monitor and select therapy in positive patients. In a breast cancer patient, for example, quarterly blood samples might be used not only to detect the presence of disease but also to re-assess ER, HER-2 and other markers, to gauge the effectiveness of therapy. It has the potential to be a key technology in transforming cancer into a chronic disease.
- Financial Impact: The financial impact could be large if the technology is clinically accepted and insurance companies reimburse appropriately for the time and effort involved. This is not a simple test to perform and interpret and requires a significant commitment in time and resources. The cost of the equipment and reagents is considerable and will probably not be purchased by small to medium sized laboratory without a significant testing volume. It should not replace any existing test, only add to it.
- Operational/Procedural Impact: All of the knowledge to run the instrument is likely to exist within most pathology labs, but the information is probably spread across several individuals. Once an area of the laboratory is selected to assume responsibility for the testing, the technicians can be easily trained. Analysis, using current systems is time consuming and requires specialized training.
- Business Model: There is not a clear business model for the pathologist. The procedure is haphazardly reimbursed and there are no CPT codes that easily map to the technologies being used. For example, the CellSearch assay employs immunomagnetic separation and image analysis on immunofluorescently stained slides, but there are no direct CPT codes for those procedures. There is no doubt that with proper re-imbursement, CTC detection could have a large financial benefit for pathologists.
Acceleration/Deceleration Triggers to Adoption
Numerous forces will drive adoption of CTCs including:
- The CellSearch assay currently requires a significant capital outlay with expensive reagent kits, but these costs are likely to shrink with competition.
- Reimbursement is unclear, and there will be no incentive to perform this assay unless re-imbursement compensates for the cost and time spent.
- Clinical data is still immature and it is still unclear whether this assay will improve patient outcome relative to other prognostic and predictive tests.s
The TAC would like to thank Chung-Che (Jeff) Chang, MD, PhD, FCAP for critical review and suggestions for the paper.
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Technology Assessment Committee (TAC) Members at the time of original POET publication:
John W. Turner, MD, FCAP, Chair
Frederick L. Baehner, MD, FCAP
Kenneth J. Bloom, MD, FCAP
Samuel K. Caughron, MD, FCAP
Thomas J. Cooper, MD, FCAP
Richard C. Friedberg, MD, PhD, FCAP
Jonhan Ho, MD, FCAP
Federico A. Monzon, MD, FCAP
David C. Wilbur, MD, FCAP
Crystal Palmatier Jenkins, MD, Junior Member
This POET was developed by the Technology Assessment Committee (TAC) with input from the Council on Scientific Affairs. Opinions expressed herein are solely those of the authors and do not represent those of the College of American Pathologists (CAP). No endorsement of any proprietary technology or product referenced is implied by the TAC or CAP. This report is provided for educational purposes only. None of the contents of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means (electronic, mechanical, photocopying, recording, or otherwise) without prior written permission of the publisher.